Detection of pathogen foodborne disease bacteria Staphylococcus aureus from German Cockroach (Blattella germanica) in the hospital area

The German cockroach (Blattella germanica) is a pest often found in apartments, houses, food-handling areas, hospitals, and healthcare facilities in Indonesia. It searches for food in dirty places such as trash cans, sewers, and septic tanks. The cockroach will feed on almost anything, including fecal matter and food for humans. So, this habit causes the German cockroach can act as a vector for several pathogenic bacteria and increases the risk of various health problems, such as foodborne diseases. One of the bacteria that cause foodborne disease is Staphylococcus aureus because these bacteria can produce toxins that cause intoxication in food. So the purpose of this study was to detect the presence of Staphylococcus aureus on cockroaches’ body surface and digestive tract. The sample of cockroaches was collected by jar trap from a hospital environment in Purwokerto (Central Java, Indonesia). The bacteria isolates were confirmed by selective medium to ensure that the isolated bacteria were S. aureus bacteria. The results showed that 26 isolates were successfully isolated from the body surface, and 21 isolates were successfully isolated from the digestive tract of the German cockroach. From these isolates, it was found that 11.53% of the isolates came from the body surface of the German cockroach, and 14.28% of the bacterial isolates isolated from the digestive tract of the German cockroach were S. aureus. The results of this study indicate that the German cockroach can be a vector for the spread of the pathogen S. aureus.


Introduction
The German cockroach (Blattella germanica) is one of the most common pests found in homes, apartments, and public facilities such as hospitals.German cockroaches like moist, warm, dark, and dirty places.These can be crevices around landfills, kitchens, warehouses, cupboards, toilets, and septic tanks.Cockroaches are known as unclean insects because they habitually regurgitate food that has entered their digestive tract [1].This behavior causes food contamination by microorganisms from digestion which can contaminate food and cause poison or disease if humans eat the food.Food that microbes have damaged can be dangerous for other fresh food [2].Each hospital has a kitchen that provides food for patients and hospital employees.German cockroaches in the hospital significantly contribute to foodborne disease outbreaks because it is used as a vector for pathogenic bacteria [3].
Cockroaches become vectors of mechanical foodborne diseases by being physically infected upon contact with fecal matter or other biohazards infested with foodborne pathogens and then physically carrying these pathogens [3].These microorganisms can be carried to the cockroach cuticle due to physical contact or in the midgut area due to the cockroach's habit of regurgitating digested food from the digestive tract [4,5].Several reports have found that the isolated microorganisms from the German cockroach show a variety of pathogenic and non-pathogenic bacteria, protozoa, fungi, and viruses [6].Several species of bacteria that are important for public health have been isolated from the surface of the bodies and digestive tract of German cockroaches, such as P. aeruginosa, S. aureus, Klebsiella pneumoniae, Citrobacter freundii, Enterobacter cloacae, E. aerogenes, Enterobacter sp., and Serratia marcescens [7].
One of the bacteria often found in B. germanica was S. aureus.This bacterium is a commensal and opportunistic pathogenic bacterium that can cause a broad spectrum of clinical infections, such as skin and soft tissue infection, bacteremia, infective endocarditis, pleuropulmonary infection, and devicerelated infections [8,9].S. aureus can survive in potentially dry and stressful environments, such as the human nose and on the skin and surfaces of things [10].These characteristics support the presence and growth of S. aureus in many foodstuffs and products and can cause foodborne disease.The toxin produced by S. aureus is thermostable and resistant to digestive enzymes, so it can cause sudden vomiting, diarrhea, nausea, malaise, stomach cramps, and pain [11].
The existence of the German cockroach in the hospital is one of the problems that must be considered because cockroaches can cause damage to food products and become disease vectors for humans or foodborne diseases.Research on bacteria S. aureus in German cockroaches has not been widely reported, whereas knowing the presence of microorganisms in them can indicate how dangerous it is and how to deal with cases of foodborne diseases.This study aimed to determine the presence of S. aureus, a microorganism that causes foodborne disease, in the body's surface and the digestive tract of German cockroaches in hospitals.

Sample preparation and isolation of bacteria from German Cockroaches
The German cockroaches were collected by jar trap from a hospital environment in Purwokerto (Central Java, Indonesia).Each trapped German cockroach was frozen at 0°C for 5-10 minutes.Then, immobilized cockroaches were added to the sterile physiological saline tube and shaken vigorously to dislodge bacteria from their body surfaces.Then, the aliquot was taken as the body surface homogenate sample and checked for bacteria growth on Nutrient Agar (Oxoid, UK).Subsequently, wash the cockroach's body with 70% alcohol for 2 minutes to remove the external contamination.After that, the body was rewashed with sterile aquadest for 2-3 minutes to remove the alcohol.The alimentary cockroach tract was dissected and macerated in mortar and pastel under sterile conditions.The homogenous suspension was prepared in sterile physiological saline.
The internal and body surface homogenate samples were diluted using the serial dilutions method.Furthermore, as much as 1 ml of sample was taken from serial dilutions 10-4, 10-5, and 10-6, then inoculated the sample using the pour plate method on Nutrient Agar (NA) (Oxoid, UK).NA media was incubated for 48 hours at room temperature.Bacterial colonies showing different morphological characters were purified by the quadrant streak method.

Micromorphological characterization of isolate
Isolates bacteria were grown on NA medium and incubated 48 hours at room temperature.Then characterized by Gram staining.One loop of the isolate was taken to be characterized micromorphologically to see the character of the cell shape and cell wall characteristics using the Gram staining technique.

Catalase test
The catalase test carried out the ability of the isolates to produce catalase.One loop of bacterial isolates was smeared on an object glass using a loop needle.The H2O2 reagent is then dripped over the bacterial isolate.A positive interpretation is indicated by the presence of gas bubbles.

Confirmed test of Staphylococcus aureus using selective medium
Isolates were incubated at 37°C for 24-48 hours on Nutrient Agar (NA) medium.The gram-positive isolate was grown on a Mannitol Salt Agar (MSA) medium and tested with a DNAse test.
Mannitol Salt Agar (MSA) medium is used to confirm mannitol fermentation.One loop isolate was streaked in MSA medium (Himedia, India) and incubated for 24 hours at 37°C.The yellow colony bacteria surrounded by a yellow zone indicated a positive result.
The DNase test was performed by incubating the isolates for 24 hours at 37°C on DNase agar (Himedia, India), and pouring an excess (~15 ml) of 1 N HCl.The clear zones around the bacterial colonies indicated DNase-positive colonies.

Data analysis
Data obtained from laboratory analysis were analyzed descriptively.The results of isolation characteristics on gram staining, catalase production ability, sugar fermentation test, mannitol fermentation on MSA media, and DNAse test were tabulated and compared with identification standards according to Bergey's Manual of Determinative Bacteriology 9th Edition [12].

and discussion
The results of bacterial isolation from German cockroaches from the hospital obtained a total of 46 isolates consisting of 26 bacterial isolates from the surface of the body and 21 bacterial isolates from the digestive tract.Almost all of the isolates obtained were gram-positive bacteria, except the GM34C isolate from the digestive tract, which was gram-negative.The 46 isolates consist of 21 isolates, including gram-positive cocci, 9 gram-positive bacilli isolates, and 17 gram-positive coccobacilli isolates (table 1).Gram staining can be used as a character in identification because it shows the difference in the outer structure of gram-positive and negative bacteria cell walls.This difference in structure results in a difference in color at the end of the gram staining procedure.The outer cell wall of gram-positive bacteria consists of thick peptidoglycan without a layer of lipoprotein or lipopolysaccharide.In contrast, gram-negative bacteria have a cell wall of thin peptidoglycan covered by a lipoprotein layer or lipopolysaccharide [13].Gram-positive bacteria have a purple color because the violet-iodine crystal dye complex is retained even when given a bleaching solution [14].Based on the results of the characteristics of several gram-positive isolates with the form of coccus, it is possible to belong to the genus Staphylococcus.Staphylococcus is a gram-positive cocci-shaped bacteria with a diameter of 0.5-1.0mm in groups, in pairs, and sometimes in short chains [15].It is necessary to test them by growing them onto a selective medium, namely MSA medium and DNAse Test medium, to ensure that the isolated bacteria are S. aureus bacteria.The DNase test is used to differentiate the activity of microorganisms based on deoxyribonuclease (DNase) and also to identify potentially pathogenic Staphylococcus bacteria.The results of this test Staphylococcus aureus gave a positive response by marking a clear zone around the colony.The clear zone formed when HCL was poured around the colony indicated that the bacteria produced the enzyme deoxyribonuclease (DNase) [16].The results of the DNAse test from 26 isolates from external bodies were 10 positive isolates, and from 20 isolates from internal bodies there were six positive isolates.These isolates showed a positive interpretation which was marked by a clear zone on the bacteria (Fig. 1).MSA medium is a differential medium for identifying bacteria that can ferment mannitol.The medium MSA test obtained 8 positive isolates from 26 isolates from external bodies and 6 isolates from 20 isolates from internal bodies (Table 2).The isolate showed a positive interpretation marked in yellow under the bacterial colony (fig.2).The combination of MSA medium and DNAse test resulted in specificity and sensitivity of 92% and 96%, respectively, and this would be the combination of choice for identifying S. aureus [17].
-+ Note : = Isolate showed positive on MSA medium and DNAse Test.
Three isolates (14.28% of total Isolate) from the body surface (GMP24A, GMP24C, and GMP35E) and three isolates (11.53% of total Isolate) from the digestive tract (GM25C, GM25F, and GM36B) showed positive results in MSA Medium and DNAse (Table 2).This Isolate was confirmed as a pathogenic S. aureus based on a selective medium.The presence of S. aureus on the external surface or digestive tract of hospital-originated cockroaches has been reported [7,18,19].The frequency of S. aureus on German cockroaches sample found in the hospital was 13.8% of the total isolate bacteria [7].It was distributed on German cockroaches' external surface body and gastrointestinal tract.Bacterial population of hospital cockroaches was higher than cockroaches obtained from residential areas due to their permanent contact with infested sites [18,20].Several bacteria isolated from hospital cockroaches showed isolates that were resistant to antibiotics, such as the MRSA strain [19,21].
Our findings represent an important public health issue regarding the presence of B. germanica in the hospital environment and its high importance as a dangerous vector for the survival and dissemination of S. aureus in the hospital environment.S. aureus in hospital cockroaches shows a dangerous effect on human health.Effective pest control measures, regular cleaning, proper food handling, and storage practices are essential to prevent the transmission of foodborne illness in hospitals and other settings.Removing cockroaches from sensitive areas, such as hospitals, is very important.Cockroaches must also be controlled in food-handling areas and patient rooms to maintain acceptable hygiene standards.

Conclusions
Based on the results of the research, conclusions can be drawn that 46 isolates were successfully isolated from the internal and external bodies of German cockroaches.A total of 14.28% of isolate bacteria from the digestive tract and 11.53% isolate from the body surface of cockroaches were confirmed as Staphylococcus aureus.

Figure 1 .
Figure 1.DNAse Test, the clear zones around the bacterial colonies indicated DNase-positive colonies.

Figure 2 .
Figure 2. MSA medium, the yellow colony bacteria surrounded by a yellow zone indicated a positive result.

Table 1 .
Characteristics of bacteria isolates.

Table 2 .
Confirmed Test Result using MSA Medium and DNAse Agar.